BB icipal Investigator/Program Director (Last, tr^^ni Messing. Robert Q. firsl^middle) DESCRIPTION. State the application's broad, long-term objectives and specific aims, making reference to the health re atedness of the project. Describe concisely the research design and methods for achieving these goals. Avoid summaries of past accomplishments and the use of the first person. This description is meant to serve as a succinct and accurate description of the proposed work when separated from the application. If Die application is funded, this description, asis,willbecomepublicinformation.Therefore,donotincludeproprietary/confidentialinformation.DONOTEXCEEDTHESPACEPROVIDED. Studies with protein kinase C epsilon (PKCE) null mice indicate that PKCe regulates anxiety and alcohol consumption, dependence and reward. This appears to be related to actions at GAB\A receptors since these mice are supersensitive to allosteric GABAA receptor agonists, including ethanol., in vivo and in vitro. The overall goal of this project is to understand how PKCe regulates GABAA receptor function. Studies will use immunohistochernistry and receptor binding autoradiography to compare the distribution of specific GABAA receptor subunits in wild type and PKce null mice: Autoradiography will also be us.ed to determine the affinity and density of binding sites for ligands that bind to benzodiazepine sites on specific: receptor subpopulations. Western analysis will be used to measure the abundance of receptor subunits in limbic brain regions that express PKCE. L(tk-) fibroblast cell lines expressing different types of receptors will be examined to determine if PKCe regulation of allosteric sensitivity is specific for a particular subunit combination. Finally a PKCe mutant that can utilize novel ATP analogs as phosphate donors will be used to identify immediate substrates of PKCe in L(tk-) cells and in synaptoneurosomes, using immunoprecipitations with antibodies to GABAA receptor subunits and associated proteins, and 2D gel electrophoresis and mass spectrometry for unknown substrates. The hope is to define a PKCe pathway to further our understanding of GABAA receptor function as it relates to alcoholism PERFORMANCE SITE(S) (organization, city, state) Ernest Gallo Clinic and Research Center, Emeryville, CA KEYPERSONNEL.SeeinstructionsonPage11.Usecontinuationpagesasneededtoprovidetherequiredinformationintheformatshownbelow. Name Organization Role on Project Robert O. Messing Ernest Gallo Clinic and Research Center Principal Investigator Kevan M. Shokat University of California, San Francisco Collaborator Maengseok Song Ernest Gallo Clinic and Research Ce:iter Postdoctoral Fellow PHS398 (Rev. 4/98) Page 2 BB Number oaaes consecutivelv at the bottom throuahout the aoDlication. Do not use suffixes such as 3a. 3b. Messing. Robert 0. Type the name of the principal investigator/progi at the top of each printed page and each uonli luaroi i page. (For type specifications, see instructions on page 6.) RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description, Performance Sites, and Personnel 2- 2 Table of Contents Detailed Budget for Initial Budget Period Budget for Entire Proposed Period of Support Budgets Pertaining to Consortium/Contractual arrangements Biographical Sketch-Principal Investigator/Program Director (Not to exceed two pages). 5-7 Other Biographical Sketches (Not to exceed two pages for each) 8-11 Other Support Resources 12 Research Plan IntroductiontoRevisedApplication(Nottoexceed3pages) Introduction to Supplemental Application (Not to exeged 25 pages). a. Specific Aims b. Background and Significance (Items a-d: not to exceed 25 pages*) c. Preliminary Studies/Progress Report.. d. Research Design and Methods 28-37 e. Human subjects 37 f. VertebrateAnimals 37-38 g. Literature Cited 38-44 h. Consortium/Contractual Arrangements i. Consultants 45 Checklist Typedarsityandtypesizeoftheenlrecpplicationmustconformblimitsprovidedininst'uctionsprovicbdininstructionsen|:ag36. Appendix (Five collated sets. No page numbering necessary for Appendix.) Check if X Appendix is Number of publications and manuscripts accepted or submitted for publication (not to exceed 10) included Other items (list):